With social development, prolongation of life expectation and change of lifestyle, diabetes mellitus has been one of the leading healthcare problems around the world. Both in developed countries and developing countries, morbidity of diabetes mellitus is rapidly growing. In 2007, there are around 246 million diabetes patients in the world, and one diabetes patient dies every 10 seconds around the world. It is estimated that the population of diabetes patients in the world will reach 333 million till 2025. China has been a severe region for diabetes. In China, the population of diabetes patients is currently 40 million, which is the second largest number in the world (the first is India), with the morbidity of around 5%. Diabetes mellitus comprises two types: one is insulin-dependent diabetes mellitus (Type 1 diabetes) and the other is non-insulin-dependent diabetes mellitus (Type 2 diabetes). Among them, Type 2 diabetes contributes to more than 90% of diabetes. Type 2 diabetes is characterized by uncontrolled insulin secretion or function as well as dysfunction of β-cell, resulting in disorder of lipid, carbohydrate and protein metabolism. It may lead to chronic hyperglycemia and eventually induce complications occur in microvasculature, macrovasculature and various organs. At present, there are two classes of medication for controlling diabetes mellitus: 1) insulin-secretion promoters, such as sulfonylureas, meglitinides, and dipeptidyl peptidase inhibitors, GLP-1 analogs; and 2) non-insulin-secretion promoters, such as insulin, α-glucosidase inhibitors, biguanides, thiazolidinediones, and insulin analogs. Currently, most anti-diabetic medications commonly used in clinic is less efficient for Type 2 diabetes. They fail to stop progressive damage of pancreas β-cell, to reduce blood level of HbA1c, and to prevent complications of diabetes, such as heart disease and kidney failure. In addition, they have, to some extent, toxic side effects. Accordingly, there is a need to study new medication for treatment of Type 2 diabetes.
In 1985, an intestinal hormone, Glucagon-like peptide-1 (GLP-1), was discovered. GLP-1 is expressed by proglucagons gene after eating, and mainly secreted by L-cells in intestinal mucosa. GLP-1 stimulates pancreas islet β-cell to secret insulin (J Med Chem, 47, 4128-4134, 2004), and play an important role in stabilizing blood glucose level. Administering GLP-1 may control blood glucose at normal level in Type 2 diabetes patients (Diabetes Care, 15, 270-276, 1992; Lancet, 359, 824-830, 2002; Endoer. Rev, 16, 390-410, 1996; Diabetologia, 28, 565-573, 1985). GLP-1 has functions as follows: glucose-dependent action on pancreas islet β-cell so as to promote the transcription of insulin gene, and increase biosynthesis and secretion of insulin; stimulating the proliferation and differentiation of β-cell and inhibiting apoptosis of β-cell so as to increase the amount of pancreas islet β-cells; inhibiting secretion of glucagons; increasing sensitivity of insulin receptors in peripheral cells; decreasing HbA1c level; suppressing appetite and eating; delaying gastric emptying (Diabetic Med, 18, 144-149, 2001; Diabetes, 51, 1443-1452, 2002; Diabetologia, 45, 1263-1273, 2002; Diabetes, 50, 525-529, 2001; Diabetes, 50, 725, 2001; Diabetes, 52, 365-371, 2003; Recent Prog. Hormne Res. 56, 377-399, 2001; Disbetologia, 39, 1546-1553, 1996; Am. J. Physicl Endocrinol. Metab, 281, E242-247, 2001; U.S. Pat. Nos. 4,779,67, 4,780,17, 4,784,25; Diabetes Care, 22, 403-408, 1999; J. Clin. Endocrinology and Metabolism, 88, 3082-3089, 2003; Diabetes, 44, 1295, 1995). However, GLP-1 is easily degraded by dipeptidyl peptidase (DPP IV) in the body, and has a half life of less than 2 minutes, and thus GLP-1 cannot be used as an effective anti-diabetic drug.
Exendin-4 is a polypeptide which is found in the saliva of the Gila monster, a poisonous lizard that lives in Arizona and New Mexico of the United States (J. Biol. Chem., 265, 20259-20262, 1990; J. Biol. Chem., 267, 7402-7405, 1992). Exendin-4 is highly homologous with GLP-1 (7-36) (53%). It is reported that Exendin-4 may bind to GLP-1 receptor and exhibits pharmacological agonistic effect similar to that of GLP-1, for example, increasing the synthesis of insulin, promoting insulin secretion in a glucose-dependent manner; stimulating proliferation and regeneration of β-cells and inhibiting apoptosis of β-cells so as to increase the amount of β-cells; inhibiting the secretion of glucagons; inhibiting generation of glycogen without inducing severe hypoglycemia; inhibiting gastrointestinal motility and secretion after eating; decreasing appetite and decreasing food uptake; protecting nerve cells (Nat. Biotech, 23, 857-861, 2005; J. Biol. Chem., 266, 2897-2902, 1991; J. Biol. Chem., 266, 21432-21437, 1992; Diabetes, 44, 16-19, 1995; Nature, 379, 69-72, 1996). The effect of Exendin-4 to promote the secretion of insulin and to inhibit the secretion of glucagon after eating is dependent on blood glucose, which is advantageous compared with currently used sulfonylureas, and in particular, Exendin-4 is less susceptible to induce hypoglycemia, and it greatly decreases the frequency of blood glucose monitoring and also lowers the body weight. A b.i.d. formulation of Exendin-4 (Exenatide, marketed as Byetta), co-developed by Amylin and Eli Lilly, is approved for market in U.S. and Europe (U.S. Pat. Nos. 5,424,286, 6,858,576, 6,872,700, 6,902,744, 6,956,026, 7,297,761). As such, this type of medication has been widely used in the treatment of diabetes and obesity around the world.
Pharmaceutical polypeptides requires multiple injections daily, since these pharmaceuticals have short half life in the body and poor physical and chemical stability, and they are susceptible to the degradation by various proteases. Exenatide is used b.i.d as an s.c. injection, which brings to patients a heavy burden in body, psychology and economy, and thus compliance of patients is limited. Accordingly, current studies of anti-diabetic drugs focus on structurally modifying Exendin-4 and developing new dosage form, in order to prolong plasma half life of Exendin-4 and increase systemic drug exposure.
Polymer modification technique is a potent modification technique which was developed since 1970s, in which PEGylation is a typical technique. This technique is to chemically conjugate PEG to a pharmaceutical protein, in order to modify the surface of the protein. Upon PEG modification, molecular weight of proteins is increased and clearance rate in kidney is decreased. In addition, a conjugated PEG chain results in steric hindrance on the modified protein molecule, leading to reduction of protein hydrolysis by proteases in blood. Thus, it efficiently prolongs the residence of proteins in circulation, and leads to prolongation of plasma half life and systemic drug exposure, resulting in improved efficacy.
At present, PEGylation has progressed into its second generation, site specific PEGylation. Site specific PEGylation can be used to specifically modify certain amino acids in proteins, and thereby prevent random modification. As such, the structure of active center in proteins is less influenced and certain antigenic sites are selectively intervened, resulting in reducing disadvantages such as reduced biological activity and increased heterogeneity due to non-site-specific PEGylation.
Scientists have carried out some studies on polymer modification of Exendin-4. Young and Prickett (CN1372570A) proved that Exendin-4 is mainly metabolized by renal clearance. Thus they modified Exendin-4 using PEG with a molecular weight ranging from 500 to 20,000 Dalton (Da). Wenchao Bao, Hongjing Xu, Gang Yu, Yajun Zuo (CN101125207A) performed amino modification in Exendin-4 using PEG with a molecular weight ranging from 20 kDa to 50 kDa.
However, there are disadvantages in existing Exendin-4 or variants thereof and various modified forms, including high administration frequency, which brings to patients heavy physical, psychological and economical burden. Thus compliance of patients is limited, and these drugs cannot be widely used. Accordingly, there still need new Exendin-4 variants and the variants modified by polymer.